The overall objectives of our research are to purify two glycolipid:glycosyltransferases from P-1798 mouse T lymphoma and to characterize the exact chemical structure (linkage) produced in the enzymatic products. Studies will also be carried out on the mode of synthesis of Ii-related hexaglycosylceramide in mouse T lymphomas (BALENTL-5 and L-4946) and B lymphomas (ABLS-140 and TEPC-15). Separation and purification of beta GalT-4 (UDP-Gal:LcOse3Cer(beta 1-4)galactosyltransferase) and beta GlcNAcT-2 (UDP-GlcNAc:nLcOse4Cer(beta 1-6)N-acetylglucosaminyltransferase will be achieved by UDP-hexitolamine-Sepharose-4B, alpha-lactalbumin-Sepharose 4B and GSL substrate-bound affinity gel column chromatography. Radioactive products from [14C-Ac]LcOse3Cer and [C63H]nLcOse4Cer will be isolated after reaction with unlabeled sugar nucleotides. The exact chemical linkages of the terminal hexose or hexosamine residues will be determined after permethylation of the purified enzymatic products and characterization by autoradiography of partially methylated radioactive units. Structure determination (after methylation) of the other glycose units will be performed by GC-mass spectrometry technique. Anomeric linkages using highly purified papaya beta-galactosidase and beta-hexosaminidase will be determined. The kinetic parameters of these two solubilized enzymes (beta GalT-4) and beta GlcNAcT-2) will be determined. Inhibition of these two enzyme activities will be studied in the presence of active site modifiers. The possible relationship between tumorigenicity and expression of Ii-related glycosphingolipid biosynthesis would be the major emphasis of this research.